There is now good evidence for the early appearance in evolution of conserved pathways for aging. These signaling pathways allow animals to postpone reproduction in unfavorable environmental conditions (Kenyon, “A Conserved Regulatory System for Aging,” Cell 105:165-168 (2001); Tissenbaum et al., “Model Organisms as a Guide to Mammalian Aging,” Dev. Cell 2:9-19 (2002)). Several elements of the signaling pathways and their related processes occur in S. cerevisiae, and provide a strong rationale for the use of yeast as a model genetic system for studying aging. For example, the phenomenon of caloric restriction is conserved as both rodents and yeast live longer when their ‘diets’ are restricted. Importantly, the genetic and molecular basis for the extended lifespan of organisms on low calorie diets is still poorly understood. In fact, it is still not known if the issue is low total calories or low carbon source, or some combination of nutrients. The conservation of these longevity mechanisms is consistent with the existence of other, yet undiscovered, mechanisms for the control and maintenance of health and/or reproductive capacity during an extended lifespan.
The effect of caloric restriction on lifespan in flies and worms is potentiated by the insulin growth factor pathway. Several homologous or analogous components of this pathway are conserved in yeast and also function in caloric restriction. The yeast protein kinase A (CRY1/PKA) signaling pathway responds to high glucose levels. CRY1/PKA mutants sporulate spontaneously in rich medium, and, significantly, increase both replicative and chronological lifespans (reviewed in Longo et al., “Evolutionary Medicine: From Dwarf Model Systems to Healthy Centenarians?” Science 299:1342-1346 (2003)). The yeast SCH9 gene is homologous to worm and mammalian Akt kinases, and also functions in yeast aging (Fabrizio et al., “Regulation of Longevity and Stress Resistance by Sch9 in Yeast,” Science 292:288-90 (2001)). These glucose-sensing pathways, when mutated or down regulated, increase lifespan. In yeast, these pathways involve nonessential components of G-protein Ras signaling (Jazwinski, “The RAS Genes: A Homeostatic Device in Saccharomyces cerevisiae Longevity,” Neurobiol. Aging 20:471-478 (1999); Jazwinski, “Growing Old: Metabolic Control and Yeast Aging,” Annu. Rev. Microbiol. 56:769-792 (2002)), and subunits of protein kinase, adenylase cyclase, and cAMP phosphodiesterase (Lin et al., “Enhanced Gluconeogenesis and Increased Energy Storage As Hallmarks of Aging in Saccharomyces cerevisiae,” J. Biol. Chem. 276:36000-36007 (2001)).
Oxidative stress has been proposed as having a significant effect on lifespan. Down regulation of glucose signaling pathways also increases oxidative stress resistance by mechanisms dependent on conserved genes, including superoxide dismutase and catalase. Superoxide dismutase and catalase normally promote growth and increase protection against oxidative stress, and the mutation of these genes in yeast decreases replicative lifespan. Tied into these pathways are genes controlling the mitochondrial retrograde response pathway (Jazwinski, “New Clues to Old Yeast,” Mech. Age. Develop. 122:865-882 (2001)). The disruption of the glucose signaling pathways in yeast increases lifespan by a SIR2-dependent mechanism (Longo et al., “Evolutionary Medicine: From Dwarf Model Systems to Healthy Centenarians?” Science 299:1342-1346 (2003)). Sir2p is a NAD+-dependent histone deacetylase that acts to regulate lifespan downstream of the glucose-response signaling pathways. The role of Sir2p in aging may be regulated by the cellular concentration of NAD+ or NADH (or their ratio), and/or nicotinamide, which rises under conditions of caloric restriction (Anderson et al., “Nicotinamide and PNC1 Govern Lifespan Extension by Calorie Restriction in Saccharomyces cerevisiae,” Nature 423:181-185 (2003); Gallo et al., “Nicotinamide Clearance by Pnc1 Directly Regulates Sir2-mediated Silencing and Longevity,” Mol. Cell. Biol. 24:1301-1312 (2004); Lin et al., “Calorie Restriction Extends Yeast Life Span by Lowering the Level of NADH,” Genes Dev. 18:12-16 (2004)).
Downstream of the primary glucose response signaling pathways are numerous effectors that affect yeast lifespan. Two examples are the yeast RecQ-like helicase genes, WRN and SGS1 (Guarente et al., “Genetic Pathways That Regulate Aging in Model Organisms,” Nature 408:255-262 (2000)). SGS1 is homologous to the human Werner syndrome gene, WRN, which causes a recessive disorder characterized by premature aging, and Bloom syndrome (BS) gene, which causes a recessive disorder characterized by short stature, and immunodeficiency. The human BS gene rescues the short lifespan of sgs mutant yeast cells, a fact which supports the conservation of lifespan mechanisms between yeast and humans (Heo et al., “Bloom's Syndrome Gene Suppresses Premature Aging Caused by Sgs1 Deficiency in Yeast Genes,” Cells 4:619-625 (1999)).
One consequence of aging in S. cerevisiae is the accumulation of extrachromosomal rDNA circles (sometime called ERCs). rDNA circles are produced by gratuitous recombination among the 100-200 tandemly repeated rDNA genes, and are maintained because they happen to contain an origin of replication. The accumulation of rDNA circles is due to age-related ‘genome instability’, a catch-all phrase for various recombination and replication errors and miscues that occur in older cells. The accumulation of rDNA circles is an especially striking and readily quantified example of age-related genomic instability (Sinclair et al., “Extrachromosomal rDNA Circles—A Cause of Aging in Yeast,” Cell 91:1033-1042 (1997)). rDNA circle accumulation requires SIR2 and is blocked by mutations in FOB1 (Guarente, “Sir2 Links Chromatin Silencing, Metabolism, and Aging,” Genes. Dev. 14:1021-1026 (2000); Jazwinski, “New Clues to Old Yeast,” Mech. Age. Develop. 122:865-882 (2001)). It is not certain that the accumulation of rDNA circles is a direct cause of aging or, alternatively, is consequence of defects in repair and replication systems that lead, for example, to increased mutation rates in older mother cells (McMurray et al., “An Age-Induced Switch to a Hyper-Recombinational State,” Science 301:1908-11 (2003)).
Lifespan in yeast has been measured in two ways, chronological age and replicative age (Jazwinski, “Metabolic Mechanisms of Yeast Aging,” Exp. Gerontol. 35:671-676 (2000)). Chronological age is the length of time that cells can survive in stationary phase, and replicative aging is the number of times a mother cell can produce a daughter cell before she senesces. Chronological and replicative aging in yeast are related, for example, because events in stationary phase affect subsequent replicative lifespan in rich medium (Longo et al., “Evolutionary Medicine: From Dwarf Model Systems to Healthy Centenarians?” Science 299:1342-1346 (2003); Ashrafi et al. “Passage Through Stationary Phase Advances Replicative Aging in Saccharomyces cerevisiae,” Proc. Natl. Acad. Sci. USA 96:9100-9105 (1999)). Although there are merits to both types of aging assays, cells nearing the end of their replicative lifespan exhibit evidence of functional senescence, which may be more relevant to metazoan aging (Jazwinski, “Growing Old: Metabolic Control and Yeast Aging,” Annu. Rev. Microbiol. 56:769-792 (2002)).
Current replicative aging assays involve counting the number of daughter cells produced by a cohort of about 40 individual mother cells placed in a grid on solid medium. This assay is tedious and usually requires close attention for 1-2 weeks. It would be desirable to identify a new assay that can quantify replicative aging in a straightforward high throughput assay.
Although, as noted above, a number of yeast genes and corresponding human homologs have been identified, there remains a need to identify other genes that affect lifespan. Moreover, to the extent that certain lifespan genes are associated with disease, it is desirable to provide an assay that can be used quickly and reliably to screen putative therapeutic agents to determine their efficacy in treating or minimizing the effects of such disease.
The present invention is directed to overcoming these and other deficiencies in the art.